Process for the production of an active molecule vector used to diffuse active substances and vector thus obtained

ABSTRACT

A process for the production of an active molecule vector that can be applied in the biomedical field, includes the following stages: 
         Diluting a monomer that has at least two NH 2  groups that are separated by at least four carbons in water, Adjusting the pH to a value of between 6.5 and 7.5,    Adding glutaraldehyde, OHC—(CH 2 ) 3 —COH, and Awaiting the polycondensation reaction and the formation of imines, and Recovering the poly(monomer-G) that is obtained. The monomer is selected from among the L-ornithine, the L-lysine or the L-citrulline. Further described are the biomedical vector that is obtained, and the use as a vector of active molecules, such as fatty acids, antioxidants, vitamin-enriched compounds or neurotransmitters for having bacteriostatic, anti-allergenic, anti-parasitic, anti-predatory or antifungal, anti-inflammatory or immunomodulating activities.

This invention relates to a process for the production of an activemolecule vector that can be applied in the biomedical field for activeingredient diffusion.

Such a vector can be applied to the diffusion of active ingredients inhuman, animal and plant kingdoms.

The invention also covers the biomedical vector that is obtained fromthis process.

In the field of treatment of the human body or the treatment of plants,for example, it is known that certain active ingredients are metabolizedprematurely before having reached their target.

Also, so that certain molecules can exhibit an adequate therapeuticactivity, it is necessary to graft these molecules onto vectors.

As active molecules that are of advantage to this invention and that aregiven by way of examples, it is possible to cite fatty acids,antioxidants, hormones, vitamin-enriched compounds, medications orneurotransmitters.

Such active molecules, exhibited by a vector, have bacteriostatic,anti-allergenic, anti-parasitic, anti-predatory or antifungal,immunomodulating or anti-inflammatory activities.

A small molecule diffuses quickly, but it is quickly metabolized whilethe same grafted molecule will have a longer service life because itwill not be metabolized as quickly.

The diffusion of an active molecule that is grafted to a suitable vectorincreases; this makes it possible to make the active ingredient migratecloser to the site of action before being metabolized, and to bestronger-acting.

The purpose is therefore to be able to use vectors with their graftedmolecules, sufficiently large in size to obtain high effectiveness butto graft them onto vectors that also assure them of high diffusibility.

Another important parameter is the capacity for the vector to receivethese active molecules by grafting.

The object of this invention is to make possible the production of apolymer-type vector that ensures this role of active molecule supportwith high diffusibility. More particularly, by modifying thepolymerization rate, this diffusibility can be adjusted.

This invention also proposes a process that makes it possible to producean active molecule vector in the form of a polymer that does not requireany inert substrate.

This same vector can also trap the heavy metals and the compounds thathave a metal that is hooked to an immune response-inducing protein suchas bovine serum albumin.

Techniques that are described in particular in Patent ApplicationPCT/FR99/00103 that make it possible to obtain polymers from amines areknown.

Diamines that are polymerized in the presence of a cross-linking agentare used for this purpose.

In these known processes, the polyamines are poly(L-ornithine-R),poly(putrescine-R), poly(cadaverine-R), poly(L-camosine-R),poly(spermidine-R) or poly(spermine-R) or else a mixture of the latter.—R represents the polymerizing agent that is reduced with sodiumborohydride.

The cross-linking agents that are used are selected from amongformaldehyde, glyoxal, malonodialdehyde, although of very high cost, orglutaraldehyde. Another agent is 1,1,3,3-tetramethoxypropane.

The polymerization process that is used consists of a dissolution of thediamine in a basic solution, beyond pH 8.0, and an addition ofglutaraldehyde.

The reduction of the double bonds is also obtained by a sodiumborohydride solution, followed by a series of dialyses.

The polymerization yield that is classified in the following order isthus obtained:poly(putrescine-G)>poly(cadaverine-G)>poly(L-ornithine-G)>poly(spermidine-G)>poly(L-camosine-G).

In these compounds, -G represents the glutaraldehyde that is reducedwith sodium borohydride.

Although the couplings of amines produced by means of glutaraldehdye areknown well, polymers that are produced with glutaraldehyde are notknown.

The problem raised by these polymers when they are used for the fluidtreatment is the necessity of working in a strongly alkaline mediumbeyond pH 8.0. The poly(putrescine-G) and the poly(L-camosine-G) cannotbe polymerized at pH levels of less than 8.0.

Such polymers are also very advantageous because it is possible togenerate three-dimensional polymers.

To produce a biomedical vector, it is not conceivable to work at a pHother than that close to neutral at 7.0, that of the human body in thiscase. It is also the same for the plant kingdom in most cases.

The purpose of this invention therefore is to determine a process thatmakes it possible to generate polymers that are two-dimensional, or,even better, three-dimensional, starting from a diamine but that work ata neutral pH or a pH that is close to this value of 7.0.

The numerous advantages of the product according to this invention willbe revealed upon reading the following description.

This process is now described in detail according to a particular,non-limiting embodiment.

The process consists in resorting to a diamine, the L-ornithine, and inpolymerizing it in the presence of a compound of the family ofdialdehydes, more particularly the glutaraldehyde, to obtain ahomopolyamine, the poly(L-omithine-G).

It is possible to carry out the same process with other diamines, evenif the yields are smaller because in the biomedical field, the necessaryquantities are smaller. It thus is possible to cite D or L-citrullineand L-lysine.

The description of this first preferred embodiment is limited toL-omithine.

This monomer comprises four carbons and two NH₂ groups. It is actuallynecessary that the two NH₂ groups be separated by at least four carbons.It is noted that tests with molecules that have three carbons do notprovide satisfaction because there is no possible polymerization.

In the case of L-omithine, it is possible to produce not only a linearhomopolymer but also a 3D homopolymer by means of a cross-linking agentthus to form a network.

The thus produced L-omithine-G homopolyamine is novel and particularlyinventive in its function as active molecule vector, more particularlyin its three-dimensional form.

The process for the production of the L-ornithine-G homopolyamineaccording to this invention consists in mixing:

the L-ornithine, for example 10 g in 25 ml of water with adjustment to apH of between 6.5 and 7.5, more particularly 7.0.NH₂—(CH₂)₃—CH(NH₂)—COOH,

glutaraldehyde, 20 ml at 50%.

OHC—(CH₂)₃—COH.

The reaction that takes place is a polycondensation reaction with imineformation.

A linear polymer is obtained that can be used by passing through adialysis system.

To obtain a 3D polymer directly, according to the process of thisinvention, a cross-linking of this polymer is ensured by adding to themedium a cross-linking agent such as polyethylenimine. The addition iscarried out in proportions of 1 ml per 10 g of omithine in this case.

The polymer that is obtained comes in the form of a three-dimensionalpolymer.

To produce beads of the homopolymer that is obtained and to make it eveneasier to manipulate, it is introduced into a hydrophobic organic mediumto obtain a two-phase effect. In addition, this medium is advantageouslyheated to further reduce the time for polymerization of the homopolymer,which becomes almost instantaneous.

To collect the beads thus formed, they are quite simply mechanicallyheld on a filter, then they are dried under a stream of hot air toeliminate the water, on the one hand, and to finalize the cross-linking,on the other hand.

These balls are next degreased and then treated at least once with soda,for example, in 200 ml of 1 M soda at 80° C. for two hours.

This stage makes it possible to remove the protons, otherwise aformation of hydrogen and a mechanical bursting of beads would takeplace, making them unsuitable for easy manipulation.

This stage can be repeated at least once.

It thus is possible to avoid unnecessarily consuming sodium borohydridesince the beads are then placed in a 1 M soda solution in the presenceof 1 g/l of sodium borohydride to reduce the double bonds of the iminesthat are formed.

The beads that are obtained are rinsed with water and 0.001 Mhydrochloric acid to neutralize possible alkaline traces and then rinsedwith copious amounts of water.

Then, L-omithine-G homopolymer beads that can be used as an activemolecule vector, with high effectiveness, are obtained. It is also notedthat it is possible to select the size of the vector and therefore thediffusibility based on the degree of cross-linking.

As an example of small molecules that can be grafted on thepoly(omithine-G), it is possible to cite the following examples:MOLECULES COUPLINGS PRODUCED CONCENTRATION (M) Palmitic AcidPalmitic-poly(ornithine-G) Ac 2.05 · 10⁻³ Myristic AcidMyristic-poly(ornithine-G) Ac 2.26 · 10⁻³ Oleic AcidOleic-poly(ornithine-G) Ac 1.96 · 10⁻³ Taurine-AGTaurine-AG-poly(ornithine-G) 3.92 · 10⁻³

The poly(L-ornithine-G) homopolyamine that is obtained by the processaccording to this invention, on which fatty acids are grafted, is alsotested from the standpoint of the toxicity, and basic tests showed anon-toxicity.

These tests consist in administering 1 mg/ml of poly(L-omithine-G)solutions grafted with fatty acids at the dose of 0.05 ml/day to malerats.

A significant weight increase is noted over the 150 days that follow.The curves are shown in an appendix to FIGS. 1 and 2.

If it is compared with L-citrulline or L-lysine, it is noted that duringthe polymerization, the yield is much lower, but a polymerization ofpoly(citrulline-G) and poly(lysine-G) is obtained with the possibilityof producing a three-dimensional polymer.

In a comparison test, 100 mg of L-omithine and 100 mg of D, L-citrullinethat are placed in the presence of 3 ml of 3 M acetate, 1 ml of waterand 3 ml of 5% glutaraldehyde are used.

The values of the weights of polymers, attained after freeze-drying, arerespectively 23.2 g of poly(omithine-G) and 7.2 mg ofpoly(citrulline-G).

This is essentially due to the CONH₂ group that reduces the availabilityfor the polymerization of the NH₂ group.

The poly(omithine-G) to which are grafted fatty acids by amide bonds wasevaluated from the standpoint of its biological activity in experimentalanimal models with chronic afflictions.

In the experimental encephalitis model, this polymer grafted with fattyacids at a concentration of 4 to 5 10⁻⁵ moles showed a biologicalactivity by ensuring significant reduction of the crisis (equivalent toa flare-up of multiple sclerosis).

1. Process for the production of an active molecule vector that can beapplied in the biomedical field, characterized in that it comprises thefollowing stages: Diluting a monomer that has at least two NH₂ groupsthat are separated by at least four carbons in water, Adjusting the pHto a value of between 6.5 and 7.5, Adding glutaraldehyde,OHC—(CH₂)₃—COH, and Awaiting the polycondensation reaction and theformation of imines, and Recovering the poly(monomer-G) that isobtained.
 2. Process for the production of an active molecule vectorthat can be applied in the biomedical field of claim 1, wherein themonomer is the L-ornithine, the L-lysine or the L-citrulline to obtainthe formation of poly(L-ornithine-G), poly(L-lysine-G), orpoly(L-citrulline-G).
 3. Process for the production of a molecule vectorthat can be applied in the biomedical field according to claim 1,wherein the polymer that is obtained is linear.
 4. Process for theproduction of a molecule vector that can be applied in the biomedicalfield according to claim 1, wherein a cross-linking agent is added toobtain a 3D network of poly(L-ornithine-G), poly(L-lysine-G), andpoly(L-citrulline-G).
 5. Process for the production of a molecule vectorthat can be applied in the biomedical field according to claim 4,wherein the cross-linking agent is polyethylenimine.
 6. Process for theproduction of a molecule vector that can be applied in the biomedicalfield according to claim 4, wherein the homopolymer that is obtained isdispersed into a hydrophobic organic medium to obtain a two-phase effectto produce beads of poly(L-ornithine-G), poly(L-lysine-G) orpoly(L-citrulline-G).
 7. Process for the production of a molecule vectorthat can be applied in the biomedical field according to claim 6,wherein to collect the thus formed beads, they are mechanically held ona filter and then dried under a stream of hot air.
 8. Process for theproduction of a molecule vector that can be applied in the biomedicalfield according to claim 6, wherein heating of the hydrophobic organicmedium that is used is initiated.
 9. Process for the production of amolecule vector that can be used in water treatment according to claim1, wherein to reduce the double bonds of the imines and to obtainamines, the following operations are initiated: Degreasing of thepolymer that is obtained resulting from the condensation reaction,Treatment at least once with soda, and Bringing this polymer into thepresence of sodium borohydride.
 10. Molecule vector that can be appliedin the biomedical field, wherein the molecule comprises thepoly(ornithine-G), the poly(L-lysine-G) or the poly(L-citrulline-G) towhich are grafted active molecules such as fatty acids, antioxidants,vitamin-enriched compounds, hormones, medications or neurotransmittersfor having bacteriostatic, anti-allergenic, anti-parasitic,anti-predatory, antifungal, anti-inflammatory or immunomodulatingactivities.
 11. A method of receiving at least one of fatty acids,antioxidants, vitamin-enriched compounds and neurotransmitters forhaving bacteriostatic, anti-allergenic, anti-parasitic, anti-predatory,antifungal, anti-inflammatory or immunomodulating activities, the methodcomprising applying an effective amount of a molecule vector comprisingat least one of the poly(ornithine-G), the poly(L-lysine-G) and thepoly(L-citrulline-G) to which are grafted active molecules selected froma group consisting of fatty acids, antioxidants, vitamin-enrichedcompounds, hormones, medications and neurotransmitters.
 12. Process forthe production of a molecule vector that can be applied in thebiomedical field according to claim 2, wherein the polymer that isobtained is linear.
 13. Process for the production of a molecule vectorthat can be applied in the biomedical field according to claim 2,wherein a cross-linking agent is added to obtain a 3D network ofpoly(L-ornithine-G), poly(L-lysine-G), and poly(L-citrulline-G). 14.Process for the production of a molecule vector that can be applied inthe biomedical field according to claim 5, wherein the homopolymer thatis obtained is dispersed into a hydrophobic organic medium to obtain atwo-phase effect to produce beads of poly(L-ornithine-G),poly(L-lysine-G) or poly(L-citrulline-G).
 15. Process for the productionof a molecule vector that can be applied in the biomedical fieldaccording to claim 7, wherein heating of the hydrophobic organic mediumthat is used is initiated.
 16. Process for the production of a moleculevector that can be used in water treatment according to claim 2, whereinto reduce the double bonds of the imines and to obtain amines, thefollowing operations are initiated: Degreasing of the polymer that isobtained resulting from the condensation reaction, Treatment at leastonce with soda, and Bringing this polymer into the presence of sodiumborohydride.
 17. Process for the production of a molecule vector thatcan be used in water treatment according to claim 3, wherein to reducethe double bonds of the imines and to obtain amines, the followingoperations are initiated: Degreasing of the polymer that is obtainedresulting from the condensation reaction, Treatment at least once withsoda, and Bringing this polymer into the presence of sodium borohydride.18. Process for the production of a molecule vector that can be used inwater treatment according to claim 4, wherein to reduce the double bondsof the imines and to obtain amines, the following operations areinitiated: Degreasing of the polymer that is obtained resulting from thecondensation reaction, Treatment at least once with soda, and Bringingthis polymer into the presence of sodium borohydride.
 19. Process forthe production of a molecule vector that can be used in water treatmentaccording to claim 5, wherein to reduce the double bonds of the iminesand to obtain amines, the following operations are initiated: Degreasingof the polymer that is obtained resulting from the condensationreaction, Treatment at least once with soda, and Bringing this polymerinto the presence of sodium borohydride.
 20. Process for the productionof a molecule vector that can be used in water treatment according toclaim 6, wherein to reduce the double bonds of the imines and to obtainamines, the following operations are initiated: Degreasing of thepolymer that is obtained resulting from the condensation reaction,Treatment at least once with soda, and Bringing this polymer into thepresence of sodium borohydride.